Indolylmaleimide derivatives

ABSTRACT

Provided are compounds of formula (I) 
                         
which are useful in the treatment and/or prevention of diseases or disorders mediated by T lymphocytes and/or PKC, e.g. acute or chronic rejection of organ or tissue allo- or xenografts, graft versus host diseases, atherosclerosis, vascular occlusion due to vascular injury such as angioplasty, restenosis, obesity, syndrome X, impaired glucose tolerance, polycystic ovary syndrome, hypertension, heart failure, chronic obstructive pulmonary disease, CNS diseases such as Alzheimer disease or amyotrophic lateral sclerosis, cancer, infectious diseases such as AIDS, septic shock or adult respiratory distress syndrome, ischemia/reperfusion injury e.g. myocardial infarction, stroke, gut ischemia, renal failure or hemorrhage shock, or traumatic shock, e.g. traumatic brain injury. The compounds of formula I are also useful in the treatment and/or prevention of T-cell mediated acute or chronic inflammatory disease or disorders or autoimmune diseases e.g. rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, Hashimotos thyroidis, multiple sclerosis, myasthenia gravis, diabetes type I or II and the disorders associated therewith, e.g. angiopathy, diabetic proliferative retinopathy, diabetic macular edema, nephropathy, neuropathy and dawn phenomenon, respiratory diseases such as asthma or inflammatory lung injury, inflammatory liver injury, inflammatory glomerular injury, cutaneous manifestations of immunologically-mediated disorders or illnesses, inflammatory and hyperproliferative skin diseases (such as psoriasis, atopic dermatitis, allergic contract dermatitis, irritant contact dermatitis and further eczematous dermatitises, seborrhoeic dermatitis), inflammatory eye diseases, e.g. Sjoegren&#39;s syndrome, keratoconjunctivitis or uveitis, inflammatory bowel disease, Crohn&#39;s disease or ulcerative colitis.

This is a continuation of application Ser. No. 10/510,027 filed on Oct.1, 2004, which is 371 of Application No. PCT/EP03/03470 filed on Apr. 2,2003, the entire disclosures of which are hereby incorporated byreference.

The present invention relates to indolylmaleimide derivatives, processfor their production and pharmaceutical compositions containing them.

More particularly the present invention provides a compound of formula I

wherein

-   R_(a) is H; CH₃; CH₂—CH₃; or isopropyl,-   R_(b) is H; halogen; C₁₋₆alkoxy; or C₁₋₆alkyl, and either

I. R is a radical of formula (a)

wherein

-   -   R₁ is piperazin-1-yl optionally substituted by CH₃ in position 3        or 4; or 4,7-diaza-spiro [2.5] oct-7-yl;    -   R₂ is Cl; Br; CF₃; or CH₃; and    -   R₃ is H; CH₃; or CF₃; R₂ being other than CH₃ or Cl when R₃ is        H, R_(a) is H or CH₃, R_(b) is    -   H and R₁ is 4-methyl-1-piperazinyl; or

II. R is a radical of formula (b)

wherein

-   -   R₄ is 1-piperazinyl substituted in positions 3 and/or 4 by CH₃;        or 4,7-diaza-spiro [2.5] oct-7-yl; R_(a) being other than H or        CH₃ when R is 4-methyl-1-piperazinyl; or

III. R is a residue of formula (c)

wherein

-   -   R₁₄ is 1-piperazinyl optionally substituted by CH₃ in position 3        and/or 4 or in position 3 by ethyl, phenyl-C₁₋₄alkyl,        C₁₋₄alkoxy-C₁₋₄alkyl or halogeno-C₁₋₄alkyl; or    -   4,7-diaza-spiro [2.5] oct-7-yl;    -   R₁₅ is halogen; CF₃; or CH₃; R₁₅ being other than CH₃ when R₁₆        is CH₃, R_(a) is H or    -   CH₃, R_(b) is H and R₁₋₄ is 4-methyl-1-piperazinyl; and    -   R₁₆ is H; CH₃; CH₂—CH₃; or CF₃; R₁₆ being other than H when R₁₅        is Cl, R_(a) is H or    -   CH₃, R_(b) is H and R₁₄ is 4-methyl-1-piperazinyl; or

IV. R is a radical of formula (d)

wherein R₈ is 1-piperazinyl, 3-methyl-piperazin-1-yl or4-benzyl-piperazin-1-yl; or

V. R is a radical of formula (e)

wherein R₉ is 4,7-diaza-spiro [2.5] oct-7-yl; or piperazin-1-ylsubstituted in position 3 by methyl or ethyl and optionally in position4 by methyl.

The compounds of formula I may exist in free form or in salt form, e.g.addition salts with e.g. organic or inorganic acids, for example,hydrochloric acid, acetic acid, trifluoroacetic acid.

It will be appreciated that the compounds of formula I may exist in theform of optical isomers, racemates or diastereoisomers. For example, aring carbon atom bearing a substituent in the position 3 of thepiperazinyl residue is asymmetric and may have the R— orS-configuration. It is to be understood that the present inventionembraces all enantiomers and their mixtures. Enantiomers are preferredover racemates. Similar considerations apply in relation to startingmaterials exhibiting asymmetric carbon atoms as mentioned.

Alkyl or alkoxy may be straight or branched. Phenyl-C₁₋₄alkyl ispreferably benzyl or phenethyl. In C₁₋₄alkoxy-C₁₋₄alkyl the alkoxymoiety is preferably methoxy or ethoxy and the alkyl moiety preferablymethyl or ethyl; a suitable example is e.g. 2-methoxyethyl. Halogen maybe F, Cl, Br or I, preferably F, Cl or Br. Halogeno-C₁₋₄alkyl is alkylwherein one or more H are replaced by halogen, e.g. Cl or F, e.g. CH₂Cl,CH₂F or CF₃

R is preferably a radical of formula (a), (c) or (e), preferably (e).

In the radical of formula (a) or (c), R₂ or R₁₅ is preferably in para toR₁ or R₁₄, respectively. R₃ is preferably in meta to R₁. In the radicalor formula (e), R₉ is preferably 4,7-diaza-spiro [2.5] oct-7-yl; when R₉is piperazinyl substituted in position 3, it has the R or Sconfiguration.

The present invention also includes a process for the preparation of acompound of formula I which process comprises reacting a compound offormula II

wherein R_(a) and R_(b) are as defined above,

-   -   with a compound of formula III        R—CH₂—CO—NH₂   (III)        wherein R is as defined above,        and, where required, converting the resulting compound of        formula I obtained in free form to a salt form or vice versa, as        appropriate.

The process may conveniently be effected in the presence of a strongbase, e.g. t-BuOK, e.g. as disclosed in WO02/38561, the contents ofwhich being incorporated herein by reference, and as illustrated in theExamples.

Compounds of formula II and III may be prepared in accordance with knownmethods, e.g. as disclosed in WO02/38561, the contents of which beingincorporated herein by reference, and as illustrated in the Examples.

Insofar as the production of the starting materials is not particularlydescribed, the compounds are known or may be prepared analogously tomethods known in the art or as described hereafter.

The following examples are illustrative of the invention without anylimitation.

-   RT=room temperature-   THF=tetrahydrofuran-   FCC=flash column chromatography-   TBAF=tetrabutyl ammonium fluoride-   BINAP=2,2′-bis(diphenylphosphino)-1,1′-binaphthyl-   Pd₂(dba)₃=Pd(0)-bis(dibenzylidenacetone)

EXAMPLE 13-[2-Chloro-3-methyl-6(4-methyl-piperazin-1-yl)-phenyl]-4-(1H-indol-3-yl)-pyrrole-2,5dione

2-[2-Chloro-3-methyl-5-(4-methyl-piperazin-1-yl)-phenyl]-acetamide (211mg, 0.75 mmol) and 3-indoleglyoxylate (270 mg, 1.35 mmol) are dissolvedin THF (5 mL). A solution of 1.0 M tert-BuOK in THF (2.98 mL, 4 eq) isadded and the mixture is stirred at 35° C. overnight. The reaction isdiluted with AcOEt (20 mL) and washed with H₂O (20 mL) and brine (10mL). The organic phase is dried over Na₂SO₄ and the solvent isevaporated. The residue is purified by FCC (AcOEt/AcOH/H₂O 7:1:1) toafford the title compound in the form of its acetate salt.

¹H NMR (DMSO, 400 MHz) δ 2.16 (s, 3H), 2.31 (s, 3H), 2.32-2.38 (m, 4H),2.97-3.10 (m, 4H), 6.61 (d, J=8.0 Hz, 1H), 6.71-6.77 (m, 2H), 7.04 (d,J=2.8 Hz, 1H), 7.08 (dd, J=7.4, 7.2 Hz, 1H), 7.41 (d, J=8.2 Hz, 1H),7.98 (d, J=1.5 Hz, 1H), 11.05 (br s, 1H), 11.90 (br s, 1H); ES-MS: 435[M+H]⁺.

2-[2-Chloro-3-methyl-5-(4-methyl-piperazin-1-yl)-phenyl]-acetamide

used as starting material may be prepared as follows:

(5Bromo-2-chloro-3-methyl-phenyl)-acetic acid methyl ester (2.0 g, 7.2mmol), N-methylpiperazine (960 μL, 8.6 mmol) and Cs₂CO₃ (3.3 g, 10.1mmol) are suspended in toluene (80 mL). Pd(OAc)₂ (81 mg, 0.36 mmol) andBINAP (224 mg, 0.36 mmol) are added and the reaction is stirred at 100°C. overnight. The mixture is filtered through Celite and the solvent isevaporated. The residue is purified by FCC (AcOEt/AcOH/H₂O 60:15:15) toafford 2-[2-chloro-3-methyl-5(4-methyl-piperazin-1-yl)-phenyl]-aceticacid methyl ester. The ester is suspended in NH₄OH 25% (60 mL). Themixture is stirred ovemight at RT and the precipitate is filtered off toyield the amide. ¹H NMR (DMSO, 400 MHz) δ 2.20 (s, 3H), 2.27 (s, 3H),2.40-2.45 (m, 4H), 3.07-3.13 (m, 4H), 3.48 (s, 2H), 6.78 (s, 1H), 6.82(s, 1H), 6.91 (br s, 1H), 7.34 (br s, 1H).

CuCl₂ (8.0 g, 0.06 mol) and tert-butylnitrile (8.9 mL, 0.074 mol) aresuspended in acetonitrile (60 mL). 1,1-dichloroethylene is addeddropwise at 20° C. 5-Bromo2-chloro-3-methyl-phenylamine (11.0 g, 0.05mol), dissolved in acetonitrile (60 mL), is added and the reaction isstirred at RT for 3 h. The mixture is poured into aqueous 20% HCl (150mL) and the aqueous phase is extracted with CH₂Cl₂ (2×150 mL). Theorganic phase is dried over Na₂SO₄ and the solvent is evaporated. Theresidue is purified by FCC (hexane/CH₂Cl₂ 9:1) to afford5-bromo-2-chloro-1-methyl-3-(2,2,2-trichloro-ethyl)-benzene. Theintermediate is redissolved in MeOH (80 mL) and heated to 70° C. Asolution of 5.4 M NaOMe in MeOH (28.4 mL) is added dropwise and thereaction is stirred at 70° C. for 3 h. The reaction is cooled to RT andconcentrated H₂SO₄ (10 mL) is added. The reaction is stirred at refluxfor 1 h. The mixture is diluted with H₂O (200 mL) and extracted withCH₂Cl₂ (2×200 mL). The combined organic phases are dried over Na₂SO₄ andthe solvent is evaporated. The residue is purified by FCC (hexane/CH₂Cl₂9:1 to 1:1) to afford (5bromo-2-chloro-3-methyl-phenyl)-acetic acidmethyl ester. ¹H NMR (CDCl₃, 400 MHz) δ 2.26 (s, 3H), 3.61 (s, 3H), 3.63(s, 2H), 7.15 (s, 1H), 7.21 (s, 1H).

By following the procedure of Example 1 but using the appropriatestarting materials, the compounds of formula I wherein R is a residue offormula (a), as indicated in Table 1 below may be obtained.

TABLE 1 Example R₁ R₂ R₃ R_(a) R_(b) M.S. Data 2-(4-methyl-piperazin-1-yl) 2-CH₃ 3-CH₃ CH₃ H MH⁺ 429 3-(4-methyl-piperazin-1-yl) 2-CH₃ 3-CH₃ H H MH⁺ 415 4-(4-methyl-piperazin-1-yl) 2-Cl 3-CH₃ CH₃ H MH⁺ 449 5 1-piperazinyl 2-Cl3-CH₃ H H MH⁺ 421 6 1-piperazinyl 2-Cl 3-CH₃ CH₃ H MH⁺ 435 73-R-methyl-piperazin-1-yl 2-Cl 3-CH₃ CH₃ H MH⁺ 449 83-R-methyl-piperazin-1-yl 2-Cl 3-CH₃ H H MH⁺ 435 9 1-piperazinyl 2-Cl3-CF₃ CH₃ H MH⁺ 503 10 1-piperazinyl 2-Cl 3-CF₃ H H MH⁺ 489 11-(4-methyl-piperazin-1-yl) 2-Cl 3-CH₃ H CH(CH₃)₂ MH⁺ 477 12-(4-methyl-piperazin-1-yl) 2-Cl 3-CH₃ H CH₃ MH⁺ 449 13-(4-methyl-piperazin-1-yl) 2-Cl 3-CH₃ H CH₂—CH₃ MH⁺ 463 14-(4-methyl-piperazin-1-yl) 2-Cl 3-CH₃ H Cl MH⁺ 469 15-(4-methyl-piperazin-1-yl) 2-Cl 3-CH₃ H F MH⁺ 453 16-(4,7-diaza-spiro[2.5]oct-7-yl) 2-Cl H H CH₂—CH₃ MH⁺ 462 17-(4,7-diaza-spiro[2.5]oct-7-yl) 2-Cl H H Cl MH⁺ 468 18-(4,7-diaza-spiro[2.5]oct-7-yl) 2-Cl H H CH₃ MH⁺ 447 19-(4,7-diaza-spiro[2.5]oct-7-yl) 2-Cl H H H MH⁺ 434

EXAMPLE 20 3-[3-(4,7-Diaza-spiro[2.5]oct-7-yl)-naphthalen-1-yl]-4-(1-methyl-1H-indol-3-yl)-pyrrole-2,5-dione

2-[3-(4,7-Diaza-spiro[2.5] oct-7-yl)-naphthalen-1-yl]-acetamide (100 mg,0.30 mmol) and (1-methyl-1H-indol-3-yl)-oxo-acetic acid methyl ester (97mg, 0.44 mmol) are azeotroped three times with dry THF and thendissolved in dry THF (3 ml). A solution of 1.0 M KOtBu in THF (1.2 ml)is added dropwise over 20 minutes at RT. After 5 minutes, TLC analysisindicates complete conversion of starting materials. The reaction isquenched by the additon of water (5 ml). The mixture is diluted withEtOAc and washed twice with saturated aq. NH₄Cl. The aqueous layers areback extracted twice with EtOAc. The combined organic layers are driedover Na₂SO₄ and the solvent is evaporated. The residue is purified byFCC (EtOAc/AcOH/H₂O 800:55:45) to afford the title compound as itsacetate salt. The compound is dissolved in MeOH/TFA and the solvent isremoved to yield the title compound as its trifluoroacetate salt. ¹H NMR(DMSO, 400 MHz) δ 0.95 (M, 4H), 3.39 (br, 4H), 3.49 (br, 2H), 3.86 (s,3H), 6.16 (d, J=7.9 Hz, 1H), 6.46 (dd, J=6.6/7.9 Hz, 1H), 7.00 (dd,J=6.6/7.9 Hz, 1H), 7.13 (dd, J=7.6/7.6 Hz, 1H), 7.40 (m, 4H), 7.55 (d,J=8.5 Hz, 1 H), 7.78 (d, J=7.6 Hz, 1H), 8.14 (s, 1H), 9.10 (br, 2H),11.17 (s, 1H); ES-MS: 463 [M+H]⁺.

2-[3-(4,7-diaza-spiro[2.5] oct-7-yl)-naphthalen-1-yl]-acetamide, used asstarting material may be prepared as follows:

a) 2-[3-(4-Benzyl-4,7-diaza-spiro[2.5]oct-7-yl)-naphthalen-1-yl]-acetamide (280 mg, 0.73 mmol) is azeotropedtwice with a 1.25 M solution of HCl in MeOH. The residue is dissolved inEtOH (10 ml). Palladium on charcoal (10%, 77 mg) is added, and themixture is stirred under an atmosphere of hydrogen (1 atm) at RT for 14h and at 50° C. for 2 h. The mixture is filtered, and the filtrate isconcentrated. The residue is purified by FCC (EtOAc/AcOH/H₂O 750:83.68to 600:150:150) to yield the title compound containing 0.7 eq of AcOH.¹H NMR (DMSO, 400 MHz) δ 0.53 (m, 4H), 1.78 (s, AcOH), 2.93 (ddd, 2H),3.04 (s, 2H), 3.16 (ddd, 2H), 3.76 (s, 2H), 6.94 (br, 1H), 6.99 (s, 1H),7.25 (dd, 1H), 7.26 (s, 1H), 7.35 (dd, J=6.7/7.8, 1 H), 7.49 (br, 1H),7.69 (d, J=7.8, 1H), 7.88 (d, J=10.0, 1H); ES-MS: 296 [M+H]⁺.

b) 2-[3-(4-Benzyl-4,7-diaza-spiro[2.5]oct-7-yl)-naphthalen-1-yl]-acetamide [3-(4-Benzyl-4,7-diaza-spiro[2.5]oct-7-yl)-naphthalen-1-yl]-acetic acid ethyl ester (347 mg, 0.84 mmol)and formamide (126 mg, 2.80 mmol) are dissolved under an atmosphere ofargon in DMF (1 ml). The solution is heated to 105° C., and NaOMe (155μL of a 5.4 M solution in MeOH, 45 mg, 0.84 mmol) is added dropwiseduring 15 minutes. After 30 minutes at 105° C., TLC analysis indicatescomplete consumption of starting material. The reaction mixture iscooled to RT, diluted with water, and extracted with EtOAc. The EtOAclayers are washed twice with water. Removal of solvent and purificationby FCC (EtOAc/MeOH 98:2 to 96:4 to 90:10) yielded the title compound. ¹HNMR (CDCl₃, 400 MHz) δ 0.71 (ddd, 2H), 0.89 (ddd, 2H), 3.10 (ddd, 2H),3.16 (s, 2H), 3.31 (ddd, 2H), 3.93 (s, 2H), 3.99 (s, 2H), 3.99 (s, 2H),5.33 (br, 1H), 5.42 (br, 1H), 7.11 (s, 1H), 7.23 (d, J=2.0, 1H), 7.32(m, 5H), 7.37 (dd, J=6.7 Hz, 1H), 7.45 (dd, J=6.7 Hz, 1H), 7.73 (d,J=8.9 Hz, 1H), 7.85 (d, J=8.9 Hz, 1H); ES-MS: 386 [M+H]⁺.

c) [3-(4-Benzyl-4,7-diaza-spiro[2.5] oct-7-yl)-naphthalen-1-yl]-aceticacid ethyl ester

(3-Trifluoromethanesulfonyloxy-naphthalen-1-yl)-acetic acid ethyl ester(500 mg, 1.38 mmol) is dissolved under an atmosphere of argon in dry THF(10 ml). 4-Benzyl-4,7diaza-spiro[2.5]octane (325 mg, 1.61 mmol) isadded, followed by the addition of K₃PO₄ (410 mg, 1.93 mmol), Pd₂(dba)₃(62 mg, 0.069 mmol) and biphenyl-2-yl-di-tert-butyl-phosphane (21 mg,0.069 mmol). The reaction mixture is heated to 80° C. After 4 h, TLCanalysis indicates complete consumption of starting materials. Thereaction mixture is cooled to RT, filtered and concentrated. The residueis purified by FCC (hexanes/EtOAc 100:0 to 90;10 to 80:20 to 70:30 to0:100) to afford the title compound. ¹H NMR (CDCl₃, 400 MHz) δ 0.69(ddd, 2H), 0.86 (ddd, 2H), 1.24 (t, J=7.0 Hz, 3H), 3.09 (ddd, 2H), 3.19(s, 2H), 3.31 (ddd, 2H), 3.93 (s, 2H), 4.02 (s, 2H), 4.17 (q, J=7.0 Hz,2H), 7.08 (s, 1H), 7.21 (s, 1H), 7.33 (m, 6H), 7.42 (dd, J=8.8 Hz, 1H),7.71 (d, J=8.8 Hz, 1H), 7.88 (d, J=8.8 Hz, 1H); ES-MS: 415 [M+H]⁺.

d) (3-Trifluoromethanesulfonyloxy-naphthalen-1-yl)-acetic acid ethylester

(3-Hydroxy-naphthalen-1-yl)-acetic acid ethyl ester (1.67 g, 7.25 mmol)is dissolved in CH₂Cl₂ (20 ml) under an atmosphere of argon. Pyridine(1.17 ml, 1.15 g, 14.50 mmol) is added, and the reaction mixture iscooled to 0° C., whereupon trifluoromethanesulfonic anhydride (1.79 ml,3.07 g, 10.88 mmol) is added dropwise. The reaction mixture is warmed toRT, and after 1 h a RT, TLC analysis indicates complete consumption ofstarting material. The reaction mixture is diluted with EtOAc and washedtwice with H₂O. The combined organic layers are dried over Na₂SO₄, thesolvent is removed, and the residue is purified by FCC (hexanes/EtOAc100:0 to 97:3 to 95:5 to 93:7 to 90:10) to yield the title compound. ¹HNMR (DMSO, 400 MHz) δ 1.19 (t, J=7.2 Hz, 3H), 4.11 (q, J=7.2 Hz, 2H),4.38 (s, 2H), 7.60 (d, J=3.0 Hz, 1H), 7.70 (m, 2H), 8.03 (m, 1H), 8.12(m, 2H); ES-MS: 362 [M+H]⁺.

e) (3-Hydroxy-naphthalen-1-yl)-acetic acid ethyl ester

(3-Benzyloxy-naphthalen-1-yl)-acetic acid ethyl ester (2.43 g, 7.58mmol) is dissolved in MeOH (50 ml). Palladium on charcoal (807 mg) isadded, and the reaction mixture is stirred at RT under an atmosphere ofhydrogen (1 atm) for 14 h. The reaction mixture is filtered.Concentration yields the pure title compound. ¹H NMR (DMSO, 400 MHz) δ1.18 (t, J=7.2 Hz, 3H), 4.05 (s, 2H), 4.10 (q, J=7.2 Hz, 2H), 7.04 (d,J=1.8 Hz, 1H), 7.06 (d, J=1.8, 1H), 7.30 (t, J=7.8, 1H), 7.40 (t, J=7.8,1H), 7.70 (d, J=7.8, 1H), 7.80 (d, J=7.8 Hz, 1H); ES-MS: 230 [M+H]⁺.

f) (3-Benzyloxy-naphthalen-1-yl)-acetic acid ethyl ester

3-Benzyloxy-1-bromo-naphthalene (5.64 g, 18.01 mmol) is dissolved underan atmosphere of argon in dry DMF (100 ml). Tributylstannanyl-aceticacid ethyl ester (7.47 g, 19.81 mmol) is added, as well as[bis(tri-ortho-tolyl-phosphine)]palladium (II) dichloride (2.83 g, 3.60mmol) and zinc(II) bromide (5.27 g, 23.41 mmol). The reaction mixture isheated to 80° C. for 3 h. The reaction mixture is diluted with EtOAc andwashed twice with diluted brine (back extracted). The combined organiclayers are dried over Na₂SO₄, the solvent is removed, and the residue ispurified by FCC (hexane/EtOAc 100:0 to 97.5:2.5 to 95:5 to 90:10). Theresulting oil, still containing tin residues, is stirred in a 1:1mixture of EtOAc/1N NaOH (200 ml) for 1 h. The mixture is extractedtwice with EtOAc. The combined organic layers are washed twice with H₂O(back extracted), dried over Na₂SO₄ and concentrated. The residue ispurified by FCC (hexane/EtOAc 100:0 to 97:3 to 95:5 to 93:7 to 92:8 to90:10) to yield the title compound. ¹H NMR (DMSO, 400 MHz) δ 1.18 (t,J=7.2 Hz, 3H), 4.10 (q, J=7.2 Hz, 2H), 4.11 (s, 2H), 5.25 (s, 2H), 7.21(d, J=3.0 Hz, 1H), 7.41 (m, 8H), 7.53 (d, J=6.6 Hz, 1H), 7.84 (dd, J=7.2Hz, 1H); ES-MS: 320 [M+H]⁺.

g) 3-Benzyloxy-1-bromo-naphthalene

4-Bromo-naphthalen-2-ol (5.0 g, 22.41 mmol) is dissolved in dry DMF (50ml) under an atmosphere of argon. Sodium hydride (986 mg of a 60%suspension in mineral oil, 592 mg, 24.65 mmol) is added and the mixtureis stirred at 50° C. for 1 h. After re-cooling to RT, benzyl bromide(3.46 ml, 4.98 g, 29.14 mmol) and tetrabutyl ammoniumiodide (828 mg,2.24 mmol) are added. After 16 h at RT, the reaction mixture is dilutedwith EtOAc. The solution is washed twice with semi-concentrated brine(back extracted). The combined organic layers are dried over Na₂SO₄, thesolvent is removed, and the residue is purified by FCC (hexane/EtOAc100:0 to 95:5 to 90:10) to yield the title compound. ¹H NMR (CDCl₃, 400MHz) δ 5.22 (s, 2H), 7.25 (d, J=1.5 Hz, 1H), 7.45 (m, 7H), 7.64 (d,J=2.4, 1H), 7.75 (d, J=7.8, 1H), 8.17 (d, J=7.8, 1H); ES-MS: 312 [M+H]⁺.

By following procedure of example 20, the compounds of formula I whereinR is a residue of formula (b), as indicated in Table 2 may be obtained.

TABLE 2 Example R₄ R_(a) M.S. Data 21 3-R-methyl-piperazin-1-yl CH₃ MH⁺452 22 -(4,7-diaza-spiro[2.5]oct-7-yl) CH₃ MH⁺ 464 23-(4,7-diaza-spiro[2.5]oct-7-yl) H MH⁺ 450 24 3-R-methyl-piperazin-1-yl HMH⁺ 438 25 3-S-methyl-piperazin-1-yl CH₃ MH⁺ 452 263-S-methyl-piperazin-1-yl H MH⁺ 438 274-methyl-3-S-methyl-piperazin-1-yl CH₃ MH⁺ 466 284-methyl-3-S-methyl-piperazin-1-yl H MH⁺ 452

By following the procedure as disclosed above or in Example 56 inWO02/38561, but using the appropriate starting materials the compoundsof formula I wherein R is a residue of formula (c), as indicated inTable 3 below may be obtained.

TABLE 3 R₁₅ R₁₆ Ex. R₁₄ in 4 in 5 R_(a) R_(b) M.S. Data 29-(4-methyl-piperazin-1-yl) Cl CH₃ H H MH⁺ 437 30-(4-methyl-piperazin-1-yl) Br H H H MH⁺ 469 31-(4-methyl-piperazin-1-yl) Br CH₃ H H MH⁺ 483 32-(4-methyl-piperazin-1-yl) Br H CH₃ H MH⁺ 483 33-(4-methyl-piperazin-1-yl) CF₃ H H H MH⁺ 457 34-(4-methyl-piperazin-1-yl) CF₃ H CH₃ H MH⁺ 471 353-R-methyl-piperazin-1-yl Cl CH₃ H H MH⁺ 437 36-(4,7-diaza-spiro[2.5]oct-7-yl) Cl CH₃ H H MH⁺ 449 37 1-piperazinyl ClCH₃ H H MH⁺ 423 38 4-methyl-3-R-methyl-piperazin-yl Cl CH₃ H H MH⁺ 45139 3-R-methoxyethyl-piperazin-1-yl Cl CH₃ H H MH⁺ 481 403-R-ethyl-piperazin-1-yl Cl CH₃ H H MH⁺ 451 41 3-R-benzyl-piperazin-1-ylCl CH₃ H H MH⁺ 514 42 3-S-methyl-piperazin-1-yl Cl CH₃ H H MH⁺ 437 434-methyl-piperazin-1-yl Cl CH₃ H CH₂—CH₂—CH₃ MH⁺ 479 443-CH₂F-piperazin-1-yl Cl CH₃ H H MH⁺ 453 45 4-methyl-piperazin-1-yl ClCH₃ H F MH⁺ 455 46 4-methyl-piperazin-1-yl Cl CH₃ H CH(CH₃)₂ MH⁺ 479 474-methyl-piperazin-1-yl Cl CH₃ H Cl MH⁺ 471 48 4-methyl-piperazin-1-ylCl CH₃ H OCH₃ MH⁺ 467 49 4-methyl-piperazin-1-yl Cl CH₃ H CH₃ MH⁺ 451 504-methyl-piperazin-1-yl Cl CH₃ H CH₂—CH₃ MH⁺ 465 514-methyl-piperazin-1-yl CF₃ H H CH₂—CH₃ MH⁺ 485 524-methyl-piperazin-1-yl CF₃ H H CH₃ MH⁺ 471 53 4-methyl-piperazin-1-yl FH H H MH⁺ 407 54 4-methyl-piperazin-1-yl F H H CH₃ MH⁺ 421 554-methyl-piperazin-1-yl F H H CH₂—CH₃ MH⁺ 435 56 4-methyl-piperazin-1-ylF CH₂—CH₃ H CH₃ MH⁺ 449 57 4-methyl-piperazin-1-yl F CH₂—CH₃ H H MH⁺ 43558 4-methyl-piperazin-1-yl F CH₂—CH₃ H CH₂—CH₃ MH⁺ 463 594-methyl-piperazin-1-yl F CH₃ H H MH⁺ 421 60 4-methyl-piperazin-1-yl FCH₃ H CH₃ MH⁺ 435 61 4-methyl-piperazin-1-yl F CH₃ H CH₂—CH₃ MH⁺ 449

By following the procedure as disclosed above or in Example 163 in WO02138561, but using the appropriate starting material the compounds offormula I wherein R is a residue of formula (d), as indicated in Table 4below, may be obtained.

TABLE 4 Example R₈ R_(a) M.S. Data 62 3-S-methyl-piperazin-1-yl CH₃ MH⁺452 63 3-R-methyl-piperazin-1-yl H MH⁺ 438 64 3-R-methyl-piperazin-1-ylCH₃ MH⁺ 452 65 4-benzyl-1-piperazinyl H MH⁺ 514 664-benzyl-1-piperazinyl CH₃ MH⁺ 528 67 1-piperazinyl CH₃ MH⁺ 438 681-piperazinyl H MH⁺ 424

EXAMPLE 69 3-[3-(4,7-Diaza-spiro[2.5]oct-7-yl)-isoquinolin-1-yl]-4-7-methyl-1H-indol-3-yl)-pyrrole-2,5-dione

2-[3-(4,7-Diaza-spiro[2.5] oct-7-yl)-isoquinolin-1-yl]-acetamide (4.95g, 16.70 mmol) and (7-Methyl-1H-indol-3-yl)-oxo-acetic acid methyl ester(5.44 g, 25.05 mmol) are azeotroped twice with dry THF. Dry THF (100 ml)is then added, and under an atmosphere of argon KOtBu (1.0 M in THF, 50ml, 50 mmol) is added dropwise during 20 min. After an additional 90min, TLC analysis indicates complete conversion of starting materials.The reaction mixture is diluted with H₂O and extracted twice with EtOAc.The combined organic layers are washed twice with saturated aq NH₄Clsolution (back extracted), dried over Na₂SO₄ and concentrated.Purification by FCC (CH₂Cl₂/MeOH 100:0 to 98:2 to 96:4 to 94:6 to 92:8to 90:10) yields the title compound, which is converted to its acetatesalt by concentration of a EtOH/AcOH solution. ¹H NMR (DMSO, 400 MHz) δ0.26-053 (br, 4 H), 1.89 (s, 3H, CH₃COOH), 2.36 (s, 3H), 2.80 (br m, 2H), 3.15-3.48 (br m, 2H), 6.14 (d, J=8.2 HZ, 1H), 6.44 (dd, J=8.2/7.4Hz, 1H), 6.75 (d, J=7.4 Hz, 1H), 7.00 (s, 1H), 7.02 (dd, J=8.2/8.2 Hz,1H), 7.40 (dd, J=8.2/8.2 Hz, 1H), 7.59 (d, J=8.2 Hz, 1H), 7.63 (d, J=8.2Hz, 1H), 7.63 (d, J=8.2 Hz, 1H), 7.97 (d, J=2.9 Hz, 1H), 11.04-11.21(br, 1H), 11.86 (d, J=2.9 Hz, 1H); ES-MS: 464 [M+H]⁺.

2-[3-(4,7-Diaza-spiro[2.5] oct-7-yl)-isoquinolin-1-yl]-acetamide used asstarting material may be prepared as follows:

a) 2-[3-(4-Benzyl-4,7-diaza-spiro[2.5]oct-7-yl)-isoquinolin-1-yl]-acetamide (490 mg, 1.27 mmol) is dissolvedin absolute MeOH (5 ml). Pd on charcoal (140 mg) is added, as well asammonium formate (200 mg, 3.17 mmol). After refluxing for 1 h (T=75°C.), an additional charge of ammonium formate (200 mg, 3.17 mmol) isadded. 1 h later, TLC analysis indicates complete conversion of thestarting material. After filtration and concentration, the residue istaken up in CH₂Cl₂ and washed with water (pH 10 by addition of 2 NNaOH). The organic layer is dried over Na₂SO₄ and the solvent isremoved. Purification by FCC (EtOAc/AcOH/H₂O 750:83:68 to 700:110:90 to650:130:120 to 600:150:150) affords the title compound as itsbis-acetate salt. ¹H NMR (DMSO, 400 MHz) δ 0.46-0.52 (m, 4H), 2.88 (t,J=5.5, 2H), 3.35 (s, 2H), 3.49 (t, J=5.5, 2H), 3.94 (s, 2H), 6.77 (s,1H), 7.00 (br s, 1H), 7.18-7.25 (m, 1H), 7.45-7.56 (m, 2H), 7.60-7.65(m, 1H), 7.95 (d, J=9.9, 1H). ES-MS: 297 [M+H]⁺.

b) 2-[3-(4-Benzyl-4,7-diaza-spiro[2.5]oct-7-yl)-isoquinolin-1-yl]-acetamide

[3-(4-Benzyl-4,7-diaza-spiro[2.5] oct-7-yl)-isoquinolin-1-yl]-aceticacid ethyl ester (700 mg, 1.68 mmol) is dissolved in dry DMF under anatmosphere of argon. Formamide (224 μl, 254 mg, 5.64 mmol) is added, andthe reaction mixture is heated to 105° C. At this temperature, NaOMe(312 μl of a 5.4 M solution in MeOH, 91 mg, 1.68 mmol) is added dropwiseduring 20 minutes. After 30 minutes at 105° C., TLC analysis indicatescomplete conversion of starting material. Cooling the reaction mixtureto RT is followed by addition of water and extraction with EtOAc. Theorganic layers are washed with H₂O (twice), dried over Na₂SO₄ andconcentrated. The residue is purified by FCC (hexanes/EtOAc 1:1 to 1:3to 0:100 to EtOAc/MeOH 98:2) to afford the title compound. ¹H NMR (DMSO,400 MHz) δ 0.68-0.70 (m, 2H), 0.82-0.88 (m, 2H), 3.08 (t, J=4.4, 2H),3.45 (s, 2H), 3.58 (t, J=4.4, 2H), 3.96 (s, 2H), 4.27 (s, 2H), 5.3-5.5(br, 1H), 6.55-6.7 (br, 1H), 6.72 (s, 1H), 7.26-7.36 (m, 6H), 7.51 (t,J=8.8, 1H), 7.60 d, J=9.9, 1H), 8.02 (d, J=9.9, 1H); ES-MS: 387 [M+H]⁺.

c) [3-(4-Benzyl-4,7-diaza-spiro[2.5] oct-7-yl)-isoquinolin-1-yl]-aceticacid ethyl ester (3-Chloro-isoquinolin-1-yl)-acetic acid ethyl ester(2.50 g, 10.01 mmol), 4-benzyl-4,7-diaza-spiro[2.5]octane (2.23 g, 11.01mmol), NaOtBu (1.06 g, 11.01 mmol), BINAP (249 mg, 0.40 mmol) andPalladium(II)-acetate (180 mg, 0.80 mmol) are mixed under argon. Afteraddition of degassed, dry dioxane (36 ml), the suspension is heated to85° C. After 25 minutes at 85° C., HPLC analysis indicates a conversionof 71%. The mixture is cooled to RT, diluted with EtOAc, and washed withH₂O and sat. aq. NH₄Cl (back extracted). The combined organic layers aredried over Na₂SO₄, the solvent is removed and the residue purified byFCC (hexane/EtOAc 100:0 to 96:4 to 93:7 to 90:10 to 85:15) to afford thetitle compound. ¹H NMR (DMSO, 400 MHz) δ 0.58-0.61 (m, 2H), 0.70-0.73(m, 2H), 1.18 (t, J=8.8, 3H), 2.98 (t, J=5.5, 2H), 3.39 (s, 2H), 3.49(t, J=5.5, 2H), 3.86 (s, 2H), 4.12 (s, 2H), 4.12 (q, J=8.8, 2H), 5.59(s, 1H), 7.14-7.19 (m, 6H), 7.39 (t, J=8.8, 1H), 7.51 (d, J=9.9, 1H),7.78 (d, J=9.9, 1H); ES-MS: 417 [M+H]⁺.

d) (3-Chloro-isoquinolin-1-yl)-acetic acid ethyl ester

1,1,1,3,3,3-Hexamethyl-disilazane (27.4 ml, 20.37 g, 126.2 mmol) aredissolved in dry toluene (150 ml). After cooling to −78° C., n-BuLi (79ml of a 1.6 M solution in hexanes, 126.2 mmol) is slowly added during 20minutes. The white suspension is stirred at −78° C. for 15 minutes andat RT for 15 minutes, after which time a clear bright yellow solution isobtained. This solution is canulated into a second two-necked flask,containing Pd₂(dba)₃ (1.39 g, 1.51 mmol) and(2′-Dicyclohexylphosphanyl-biphenyl-2-yl)-dimethyl-amine (1.25 g, 3.18mmol). After stirring at RT for 10 minutes, the clear dark red solutionis cooled to −10° C. Acetic acid tert-butyl ester (15.7 ml, 13.5 g,116.1 mmol) is added during 5 min. After 10 minutes at −10° C.1,3-Dichloro-isoquinoline (10.0 g, 50.49 mmol) is added in one portion.The dark red solution is allowed to warm to RT. After 30 minutes at RT,TLC analysis indicates complete conversion of the starting material. Thereaction mixture is filtered through a 2-cm pad of silica, which isrinsed with EtOAc/MeOH 98:2. After concentration, the residue ispurified by FCC (toluene/CH₂Cl₂ 2:1 to toluene/EtOAc 100:0 to 99:1 to98:2 to 97:3 to 96:4 to 94:6 to 90:10) to afford(3-Chloro-isoquinolin-1-yl)-acetic acid tert.-butyl ester. This compoundis dissolved in a saturated ethanolic solution of HCl (200 ml) andrefluxed for 15 minutes. Concentration affords the title compound inquantitative yield. ¹H NMR (DMSO, 400 MHz) δ 1.17 (t, J=8.8, 3H), 4.11(q, J=8.8, 2H), 4.28 (s, 2H), 7.51-7.57 (m, 1H), 7.61 (s, 1H), 7.61-7.66(m, 1H), 7.72 (d, J=8.8, 1H), 7.98 (d, J=8.8, 1H); ES-MS: 250 [M+H]⁺.

By following the procedure of Example 69 but using the appropriatestarting materials, the compounds of formula I wherein R is residue offormula (e), as indicated in Table 5 below, may be obtained.

TABLE 5 Example R₉ R_(a) R_(b) M.S. Data 70-(4,7-diaza-spiro[2.5]oct-7-yl) CH₃ H MH⁺ 465 71 3-ethyl-piperazin-1-ylCH₃ H MH⁺ 467 72 -(4,7-diaza-spiro[2.5]oct-7-yl) H H MH⁺ 451 733-ethyl-1-piperazinyl H H MH⁺ 453 74 3-R-methyl-piperazin-1-yl CH₃ H MH⁺453 75 3-R-methyl-piperazin-1-yl H H MH⁺ 439 763-S-methyl-piperazin-1-yl CH₃ H MH⁺ 453 77 3-S-methyl-piperazin-1-yl H HMH⁺ 439 78 4-methyl-3-S-methyl-piperazin-1-yl CH₃ H MH⁺ 467 794-methyl-3-S-methyl-piperazin-1-yl H H MH⁺ 453  80*4,7-diaza-spiro[2.5]7ct-7-yl CH₃ H MH⁺ 464 814,7-diaza-spiro[2.5]7ct-7-yl H F MH⁺ 479 82 4,7-diaza-spiro[2.5]7ct-7-ylCH₂—CH₃ H MH⁺ 479 83 4,7-diaza-spiro[2.5]7ct-7-yl H CH(CH₃)₂ MH⁺ 495 844,7-diaza-spiro[2.5]7ct-7-yl H OCH₃ MH⁺ 471 854,7-diaza-spiro[2.5]7ct-7-yl CH₃ CH₂—CH₃ MH⁺ 451 864,7-diaza-spiro[2.5]7ct-7-yl H CH₂—CH₃ MH⁺ 465 874,7-diaza-spiro[2.5]7ct-7-yl CH(CH₃)₂ H MH⁺ 451 884,7-diaza-spiro[2.5]7ct-7-yl CH₃ CH₃ MH⁺ 479 894,7-diaza-spiro[2.5]7ct-7-yl CH₃ Cl MH⁺ 499 904,7-diaza-spiro[2.5]7ct-7-yl H Cl MH⁺ 485 914,7-diaza-spiro[2.5]7ct-7-yl CH₂—CH₃ CH₃ MH⁺ 492 923-ethyl-piperazin-1-yl CH₂—CH₃ CH₂—CH₃ MH⁺ 494 *The compound of Example80 is converted into its bis-trifluoroacetate or acetate salt.

The compounds of formula I in free form or in pharmaceuticallyacceptable salt form exhibit valuable pharmacological properties, e.g.inhibiting Protein Kinase C (PKC), e.g. PKC isoforms like α, β, δ, ε, ηor θ activity, inhibiting T-cell activation and proliferation, e.g. byinhibiting production by T-cells or cytokines, e,g, IL-2, by inhibitingthe proliferative response of T-cells to cytokines, e.g. IL-2, e.g. asindicated in in vitro and in vivo tests and are therefore indicated fortherapy.

A. In Vitro

1. Protein Kinase C Assay

The compounds of formula I are tested for their activity on differentPKC isoforms according to a published method (D. Geiges et al. Biochem.Pharmacol. 1997;53:865-875) The assay is performed in a 96-wellpolypropylene microtiterplate (Costar 3794) that has been previouslysiliconized with Sigmacote (Sigma SL-2). The reaction mixture (50 μl)contains 10 μl of the relevant PKC isozyme together with 25 μl of thetest compound and 15 μl of a mix solution that contains 200 μg/mlprotamine sulfate, 10 mM Mg(NO₃)₂, 10 μM ATP (Boehringer 519987) and3750 Bq of ³³P-ATP (Hartmann Analytic SFC301, 110TBq/mmol) in 20 mMTris-buffer pH 7.4+0.1% BSA. Incubation is performed for 15 min. at 32°C. in a microtiterplate shaking incubator (Biolabo ScientificInstruments). Reaction is stopped by adding 10 μl of 0.5 M Na₂EDTA, pH7.4. 50 μl of mixture are pipetted onto a pre-wetted phosphocellulosepaper (Whatmann 3698-915) under gentle pressure. Non-incorporated ATP iswashed away with 100 μl bi-dist H₂O. The paper is washed twice in 0.5%H₃PO₄ for 15 min followed by 5 min in EtOH. Thereafter the paper isdryed and placed in an omnifilter (Packard 6005219), and overlayed with10 μl/well of Microscint-O (Packard 6013611) before counting in aTopcount radioactivity counter (Packard). IC₅₀ measurement is performedon a routine basis by incubating a serial dilution of inhibitor atconcentrations ranging between 1-1000 μM according to the methoddescribed above. IC₅₀ values are calculated from the graph by sigmoidalcurve fitting.

2. Protein Kinase C θ Assay

Human recombinant PKCθ is used under the assay conditions as describedabove. In this assay, compounds of formula I inhibit PKC θ with anIC₅₀≦1 μM. Compound of Examples 33 and 69 inhibit PKCθ in this assaywith an IC₅₀ 6.8 and 12.1 nM, respectively.

3. Protein Kinase Cα Assay

Human recombinant PKCα was obtained from Oxford Biomedical Research andis used under the assay conditions as described under Section A.1 above.In this assay, compounds of formula I inhibit PKC θ with an IC₅₀≦1 μM.Compound of Example 29 inhibits PKCα in this assay with an IC₅₀ of 4.3nM.

4. Protein Kinase Cβ1 Assay

Human recombinant PKCβ1 was obtained from Oxford Biomedical Research andis used under the assay conditions as described under Section A.1 above.In this assay, compounds of formula I inhibit PKC θ with an IC₅₀≦1 μM.Compound of Example 33 inhibits PKCβ1 in this assay with an IC₅₀ of 19.6nM.

5. Protein Kinase Cδ Assay

Human recombinant PKCδ was obtained from Oxford Biomedical Research andis used under the assay conditions as described under Section A.1 above.In this assay, compounds of formula I inhibit PKC θ with an IC₅₀≦1 μM.Compound of Example 29 inhibits PKCδ in this assay with an IC₅₀ of 20nM.

6. Protein Kinase Cε Assay

Human recombinant PKCε was obtained from Oxford Biomedical Research andis used under the assay conditions as described under Section A.1 above.In this assay, compounds of formula I inhibit PKC θ with an IC₅₀≦1 μM.Compound of Example 69 inhibits PKCε in this assay with an IC₅₀ of 18nM.

7. Protein Kinase Cη Assay

Human recombinant PKCη was obtained from PanVera and is used under theassay conditions as described under Section A.1 above. In this assay,compounds of formula I inhibit PKC θ with an IC₅₀≦1 μM. Compound ofExample 29 inhibits PKCη in this assay with an IC₅₀ of 27.4 nM.

8. CD28 Costimulation Assay

The assay is performed with Jurkat cells transfected with a humaninterleukin-2 promoter/reporter gene construct as described by Baumann Get al. in Transplant. Proc. 1992;24:43-8, the β-galactosidase reportergene being replaced by the luciferase gene (de Wet J., et al., Mol. CellBiol. 1987, 7(2), 725-737). Cells are stimulated by solid phase-coupledantibodies or phorbol myristate acetate (PMA) and the Ca⁺⁺ ionophoreionomycin as follows. For antibody-mediated stimulation Microlite TM1microtiter plates (Dynatech) are coated with 3 μg/ml goat anti-mouse IgGFc antibodies (Jackson) in 55 μl phosphate-buffered saline (PBS) perwell for three hours at RT. Plates are blocked after removing theantibodies by incubation with 2% bovine serum albumin (BSA) in PBS (300μl per well) for 2 hours at RT. After washing three times with 300 μlPBS per well, 10 ng/ml anti-T cell receptor antibodies (WT31, Becton &Dickinson) and 300 ng/ml anti-CD28 antibodies (15E8) in 50 μl 2% BSA/PBSare added as stimulating antibodies and incubated overnight at 4° C.Finally the plates are washed three times with 300 μl PBS per well.Seven three-fold serial dilutions of test compounds in duplicates inassay medium (RPMI 1640/10% fetal calf serum (FCS) containing 50 μM2-mercaptoethanol, 100 units/ml penicillin and 100 μg/ml streptomycin)are prepared in separate plates, mixed with transfected Jurkat cells(clone K22 290_H23) and incubated for 30 minutes at 37° C. in 5% CO₂.100 μl of this mixture containing 1×10⁵ cells are then transferred tothe antibody-coated assay plates. In parallel 100 μl are incubated with40 ng/ml PMA and 2 μM ionomycin. After incubation for 5.5 hours at 37°C. in 5% CO₂, the level of luciferase is determined by bioluminescencemeasurement. The plates are centrifuged for 10 min at 500 g and thesupernatant is removed by flicking. Lysis buffer containing 25 mMTris-phosphate, pH 7.8, 2 mM DTT, 2 mM1.2-diaminocyclohexane-N,N,N′,N-tetraacetic acid, 10% (v/v) glycerol and1% (v/v) Triton X-100 is added-(20 μl per well). The plates areincubated at RT for 10 minutes under constant shaking. Luciferaseactivity is assessed with a bioluminescence reader (Labsystem, Helsinki,Finland) after automatic addition of 50 μl per well luciferase reactionbuffer containing 20 mM Tricine, 1.07 mM (MgCO₃)₄Mg(OH)₂×5H₂O, 2.67 mMMgSO₄, 0.1 mM EDTA, 33.3 mM DTT, 270 μM coenzyme A, 470 μM luciferin(Chemie Brunschwig AG), 530 μM ATP, pH 7.8. Lag time is 0.5 seconds,total measuring time is 1 or 2 seconds. Low control values are lightunits from anti-T cell receptor- or PMA-stimulated cells, high controlsare from anti-T cell receptor/anti-CD28- or PMA/ionomycin-stimulatedcells without any test sample. Low controls are subtracted from allvalues. The inhibition obtained in the presence of a test compound iscalculated as percent inhibition of the high control. The concentrationof test compounds resulting in 50% inhibition (IC₅₀) is determined fromthe dose-response curves. In this assay, compounds of formula I inhibitanti-T cell receptor/anti-CD28 and PMA/ionomycin stimulated Jurkat cellswith an IC₅₀≦1 μM. Compound of Example 29 has an IC₅₀ of 20 nM in thisassay.

9. Allogeneic Mixed Lymphocyte Reaction (MLR)

The two-way MLR is performed according to standard procedures (J.Immunol. Methods, 1973, 2, 279 and Meo T. et al., Immunological Methods,New York, Academic Press, 1979, 227-39). Briefly, spleen cells from CBAand BALB/c mice (1.6×10⁵ cells from each strain per well in flat bottomtissue culture microtiter plates, 3.2×10⁵ in total) are incubated inRPMI medium containing 10% FCS, 100 U/ml penicillin, 100 μg/mlstreptomycin (Gibco BRL, Basel, Switzerland), 50 μM 2-mercaptoethanol(Fluka, Buchs, Switzerland) and serially diluted compounds. Seventhree-fold dilution steps in duplicates per test compound are performed.After four days of incubation 1 μCi ³H-thymidine is added. Cells areharvested after an additional five-hour incubation period, andincorporated ³H-thymidine is determined according to standardprocedures. Background values (low control) of the MLR are theproliferation of BALB/c cells alone. Low controls are subtracted fromall values. High controls without any sample are taken as 100%proliferation. Percent inhibition by the samples is calculated, and theconcentrations required for 50% inhibition (IC₅₀ values) are determined.In this assay, Compound of Example 29 has an IC₅₀ of 28 nM. Compounds offormula I also exhibit IC₅₀ values in the nM range when tested in thehuman MLR.

10. Inhibition of GSK-3β

The GSK-3β binding assay is performed in 50 μl reactions in 96 wellpolypropylene plate, each reaction containing 20 mM magnesium chloride,40 μM ATP, 2 mM DTT, 88.5 μM biotinylated and phosphorylatedCREB-peptide substrate (biotin-KRREILSRRPS(PO₄)YR—OH; Q. M. Wang et al.,J. Biol. Chem. 269, 14566-14574, 1994), [γ-³³P]ATP (1 μCi) and 2 μl ofthe compound to be tested in DMSO(various concentrations). 15 μl ofGSK-3β (various concentrations) is added and the mixture is incubated at30° C. for 1 hour. The reaction is stopped by transferring 25 μl of themixture to a phosphocellulose plate containing 130 μl of 1.85%phosphoric acid. The free radionucleotides in the membrane are washedoff under vacuum with 1.85% phosphoric acid (5 times). After the lastwash, the plate is transferred to an adaptor plate and 50 μl ofscintillation cocktail (Microscint-20, Packard, cat. a 20-133) is addedto each well and the amount of radioactivity is counted in a topcounter. Compounds of formula I are active in this assay. Compounds offormula I may also be tested in other standard GSK-3β binding assaysusing other substrates, e.g. as commercially available.

B. In Vivo

Rat Heart Transplantation

The strain combination used: Male Lewis (RT¹ haplotype) and BN (RT¹haplotype). The animals are anaesthetised using inhalationalisofluorane. Following heparinisation of the donor rat through theabdominal inferior vena cava with simultaneous exsanguination via theaorta, the chest is opened and the heart rapidly cooled. The aorta isligated and divided distal to the first branch and the brachiocephalictrunk is divided at the first bifurcation. The left pulmonary artery isligated and divided and the right side divided but left open. All othervessels are dissected free, ligated and divided and the donor heart isremoved into iced saline.

The recipient is prepared by dissection and cross-clamping of theinfra-renal abdominal aorta and vena cava. The graft is implanted withend-to-side anastomoses, using 10/0 monofilament suture, between thedonor brachiocephalic trunk and the recipient aorta and the donor rightpulmonary artery to the recipient vena cava. The clamps are removed, thegraft tethered retroabdominally, the abdominal contents washed with warmsaline and the animal is closed and allowed to recover under a heatinglamp. Graft survival is monitored by daily palpation of the beatingdonor heart through the abdominal wall. Rejection is considered to becomplete when heart beat stops. Increases of graft survival are obtainedin animals treated with a compound of formula I administered orally at adaily dose of 1 to 30 mg/kg bid.

Graft v. Host Model

Spleen cells (2×10⁷) from Wistar/F rats are injected subcutaneously intothe right hind footpad of (Wistar/F×Fischer 344)F₁ hybrid rats. The leftfootpad is left untreated. The animals are treated with the testcompounds on 4 consecutive days (0-3). The popliteal lymph nodes areremoved on day 7, and the weight differences between two correspondinglymph nodes are determined. The results are expressed as the inhibitionof lymph node enlargement (given in percent) comparing the lymph nodeweight differences in the experimental groups to the weight differencebetween the corresponding lymph nodes from a group of animals leftuntreated with a test compound. In this assay, an inhibition of 100% isobtained with compound of Example 29 when administered at a dose of 30mg/kg/day bid.

The compounds of formula I are, therefore, useful in the treatmentand/or prevention of diseases or disorders mediated by T lymphocytesand/or PKC, e.g. acute or chronic rejection of organ or tissue allo- orxenografts, graft versus host diseases, atherosclerosis, vascularocclusion due to vascular injury such as angioplasty, restenosis,obesity, syndrome X, impaired glucose tolerance, polycystic ovarysyndrome, hypertension, heart failure, chronic obstructive pulmonarydisease, CNS diseases such as Alzheimer disease or amyotrophic lateralsclerosis, cancer, infectious diseases such as AIDS, septic shock oradult respiratory distress syndrome, ischemia/reperfusion injury e.g.myocardial infarction, stroke, gut ischemia, renal failure or hemorrhageshock, or traumatic shock, e.g. traumatic brain injury. The compounds offormula I are also useful in the treatment and/or prevention of T-cellmediated acute or chronic inflammatory diseases or disorders orautoimmune diseases e.g. rheumatoid arthritis, osteoarthritis, systemiclupus erythematosus, Hashimoto's thyroidis, multiple sclerosis,myasthenia gravis, diabetes type I or II and the disorders associatedtherewith, e.g. angiopathy, diabetic proliferative retinopathy, diabeticmacular edema, nephropathy, neuropathy and dawn phenomenon, respiratorydiseases such as asthma or inflammatory lung injury, inflammatory liverinjury, inflammatory glomerular injury, cutaneous manifestations ofimmunologically-mediated disorders or illnesses, inflammatory andhyperproliferative skin diseases (such as psoriasis, atopic dermatitis,allergic contact dermatitis, irritant contact dermatitis and furthereczematous dermatitises, seborrhoeic dermatitis), inflammatory eyediseases, e.g. Sjoegren's syndrome, keratoconjunctivitis or uveitis,inflammatory bowel disease, Crohn's disease or ulcerative colitis. Forthe above uses the required dosage will of course vary depending on themode of administration, the particular condition to be treated and theeffect desired. In general, satisfactory results are indicated to beobtained systemically at daily dosages of from about 0.1 to about 100mg/kg body weight. An indicated daily dosage in the larger mammal, e.g.humans, is in the range from about 0.5 mg to about 2000 mg, convenientlyadministered, for example, in divided doses up to four times a day or inretard form.

The compounds of formula I may be administered by any conventionalroute, in particular enterally, e.g. orally, e.g. in the form of tabletsor capsules, or parenterally, e.g. in the form of injectable solutionsor suspensions, topically, e.g. in the form of lotions, gels, ointmentsor creams, or in a nasal or a suppository form. Pharmaceuticalcompositions comprising a compound of formula I in free form or inpharmaceutically acceptable salt form in association with at least onepharmaceutical acceptable carrier or diluent may be manufactured inconventional manner by mixing with a pharmaceutically acceptable carrieror diluent. Unit dosage forms for oral administration contain, forexample, from about 0.1 mg to about 500 mg of active substance.

Topical administration is e.g. to the skin. A further form of topicaladministration is to the eye.

The compounds of formula I may be administered in free form or inpharmaceutically acceptable salt form e.g. as indicated above. Suchsalts may be prepared in conventional manner and exhibit the same orderof activity as the free compounds.

In accordance with the foregoing the present invention further provides:

-   1.1 A method for preventing or treating disorders or diseases    mediated by T lymphocytes and/or PKC or GSK-3β, e.g. such as    indicated above, in a subject in need of such treatment, which    method comprises administering to said subject an effective amount    of a compound of formula I or a pharmaceutically acceptable salt    thereof;-   1.2 A method for preventing or treating acute or chronic transplant    rejection or T-cell mediated inflammatory or autoimmune diseases,    e.g. as indicated above, in a subject in need of such treatment,    which method comprises administering to said subject an effective    amount of a compound of formula I or a pharmaceutically acceptable    salt thereof;-   2. A compound of formula I, in free form or in a pharmaceutically    acceptable salt form for use as a pharmaceutical, e.g. in any of the    methods as indicated under 1.1 and 1.2 above.-   3. A pharmaceutical composition, e.g. for use in any of the methods    as in 1.1 and 1.2 above comprising a compound of formula I in free    form or pharmaceutically acceptable salt form in association with a    pharmaceutically acceptable diluent or carrier therefor.-   4. A compound of formula I or a pharmaceutically acceptable salt    thereof for use in the preparation of a pharmaceutical composition    for use in any of the method as in 1.1 and 1.2 above.

Compounds of formula I may be administered as the sole active ingredientor together with other drugs in immunomodulating regimens or otheranti-inflammatory agents e.g. for the treatment or prevention of allo-or xenograft acute or chronic rejection or inflammatory or autoimmunedisorders. For example, they may be used in combination withcyclosporines, or ascomycines or their immunosuppressive analogs orderivatives, e.g. cyclosporin A, ISA Tx247, FK-506, ABT-281, ASM 981; anmTOR inhibitor, e.g. rapamycin, 40-O-(2-hydroxy)ethyl-rapamycin, CC1779,ABT578 or a rapalog, e.g. AP23573 etc.; corticosteroids;cyclophosphamide; azathioprene; methotrexate; an EDG receptor agonisthaving accelerating lymphocyte homing properties, e.g. FTY 720 or ananalogue thereof; leflunomide or analogs thereof; mizoribine;mycophenolic acid; mycophenolate mofetil; 15-deoxyspergualine or analogsthereof; immunosuppressive monoclonal antibodies, e.g., monoclonalantibodies to leukocyte receptors, e.g., MHC, CD2, CD3, CD4, CD11a/CD18, CD7, CD25, CD 27, B7, CD40, CD45, CD58, CD 137, ICOS, CD150(SLAM), OX40, 4-1BB or their ligands, e.g. CD154; or otherimmunomodulatory compounds, e.g. a recombinant binding molecule havingat least a portion of the extracellular domain of CTLA4 or a mutantthereof, e.g. an at least extracellular portion of CTLA4 or a mutantthereof joined to a non-CTLA4 protein sequence, e.g. CTLA41g (for ex.designated ATCC 68629) or a mutant thereof, e.g. LEA29Y, or otheradhesion molecule inhibitors, e.g. mAbs or low molecular weightinhibitors including LFA-1 antagonists, Selectin antagonists and VLA-4antagonists. Compounds of formula I may also be administered togetherwith an antiproliferative drug, e.g. a chemotherapeutic drug, e.g. asused in cancer treatment, including but not limited to aromataseinhibitors, antiestrogens, topoisomerase I inhibitors, topoisomerase IIinhibitors, microtubule active agents, alkylating agents, histonedeacetylase inhibitors, farnesyl transferase inhibitors, COX-2inhibitors, MMP inhibitors, mTOR inhibitors, antineoplasticantimetabolites, platin compounds, compounds decreasing the proteinkinase activity and further anti-angiogenic compounds, gonadorelinagonists, anti-androgens, bengamides, bisphosphonates, antiproliferativeantibodies and temozolomide, or with an anti-diabetic drug, an insulinsecretagogue or insulin secretion enhancer, e.g. a sulphonyl urea, e.g.tolbutamide, chlorpropamide, tolazamide, acetohexamide,4-chloro-N-[(1-pyrolidinylamino)carbonyl]-benzensulfonamide(glycopyramide), glibenclamide (glyburide), gliclazide,1-butyl-3-metanilylurea, carbutamide, glibonuride, glipizide,gliquidone, glisoxepid, glybuthiazole, glibuzole, glyhexamide,glymidine, glypinamide, phenbutamide or tolylcyclamide, an oralinsulinotropic agent derivative, e.g. a short acting insulin enhancer,e.g. meglitinide, repaglinide, a phenyl acetic acid derivative,e.g.nateglinide, a DPP IV inhibitor, e.g.1-{2-[(5-cyanopyrdin-2-yl)amino]ethylamino}acetyl-(2S)-cyano-pyrrolidinedihydrochloride, LAF237, GLP-1 or a GLP-1 agonist analog, or an insulinsensitizer e.g. a peroxisome proliferator activated receptor γ agonist(PPARγ), e.g. a glitazone, a non-glitazone type such as aN-(2-benzoylphenyl)-L-tyrosine analogue, e.g. GI-262570, or anoxolidinedione, e.g. JTT501, a dual PPARγ/PPARα agonist, e.g.DRF-554158, NC-2100 or NN-622, a retinoid X receptor agonist or arexinoid, e.g.2-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-cyclopropyl]-pyridine-5-carboxylicacid,4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-2-carbonyl]-benzoicacid, 9-cis retinoic acid or an analog, derivative or a pharmaceuticallyacceptable salt thereof, in diabetes therapy,

In accordance with the foregoing the present invention provides in a yetfurther aspect:

-   5. A method as defined above comprising co-administration, e.g.    concomitantly or in sequence, of a therapeutically effective amount    of an inhibitor of GSK-3β, PKC or of T-cell activation and    proliferation, e.g. a compound of formula I in free form or in    pharmaceutically acceptable salt form, and a second drug substance,    said second drug substance being an immunosuppressant,    immunomodulatory, anti-inflammatory, antiproliferative or    anti-diabetic drug, e.g. as indicated above.-   6. A therapeutic combination, e.g. a kit, comprising a) an inhibitor    of GSK-3β, PKC or of T-cell activation and proliferation, e.g. a    compound of formula I in free form or in pharmaceutically acceptable    salt form, and b) at least one second agent selected from an    immunosuppressant, immunomodulatory, anti-inflammatory,    antiproliferative and anti-diabetic drug. Component a) and    component b) may be used concomitantly or in sequence. The kit may    comprise instructions for its administration.

Where an inhibitor of GSK-3β, PKC or of T-cell activation andproliferation, e.g. a compound of formula I, is administered inconjunction with other immunosuppressive/immunomodulatory,anti-inflammatory, antiproliferative or anti-diabetic therapy, e.g. forpreventing or treating acute or chronic graft rejection or inflammatoryor autoimmune disorders as hereinabove specified, dosages of theco-administered immunosuppressant, immunomodulatory, anti-inflammatory,antiproliferative or anti-diabetic compound will of course varydepending on the type of co-drug employed, e.g. whether it is a steroidor a cyclosporine, on the specific drug employed, on the condition beingtreated and so forth.

Compounds of formula I have an interesting pharmacokinetic profile andinteresting in vitro and in vivo activities.

1. A method for treating acute or chronic rejection of organ or tissueallo- or xenografts or graft versus host disease in a subject in need ofsuch treatment, which method comprises administering to said subject aneffective amount of a compound of formula I

wherein R_(a) is H; CH₃; CH₂-CH₃; or isopropyl, R_(b) is H; halogen;C₁₋₆alkoxy; or C₁₋₆alkyl, and either I. R is a radical of formula (a)

wherein R₁ is piperazin-1-yl optionally substituted by CH₃ in position 3or 4; or 4,7-diaza-spiro [2.5] oct-7-yl; R₂ is Cl; Br; CF₃; or CH₃; andR₃ is H; CH₃; or CF₃; R₂ being other than CH₃ or Cl when R₃ is H, R_(a)is H or CH₃, R_(b) is H and R₁ is 4-methyl-1-piperazinyl; or II. R is aradical of formula (b)

wherein R₄ is piperazin-1-yl substituted in positions 3 and/or 4 by CH₃;or 4,7-diaza-spiro [2.5] oct-7-yl; R_(a) being other than H or CH₃ whenR₄ is 4-methyl-1-piperazinyl; or II. R is a residue of formula (c)

wherein R₁₄ is 4,7-diaza-spiro [2.5] oct-7-yl; R₁₅ is halogen; CF₃; orCH₃; R₁₅ being other than CH₃ when R₁₆ is CH₃, R_(a) is H or CH₃, andR_(b) is H; and R₁₆ is H; CH₃; CH₂-CH₃; or CF₃; R₁₆ being other than Hwhen R₁₅ is Cl, R_(a) is H or CH₃, and R_(b) is H; or IV. R is a radicalof formula (d)

wherein R₈ is 1-piperazinyl, 3-methyl-piperazin-1-yl or4-benzyl-piperazin-1-yl; or V. R is a radical of formula (e)

wherein R₉ is 4,7-diaza-spiro [2.5] oct-7-yl; or 1-piperazinylsubstituted in position 3 by methyl or ethyl and optionally in position4 by methyl; or a pharmaceutically acceptable salt thereof.
 2. A methodaccording to claim 1 wherein the compound of formula I is administeredby topical route to the skin or to the eye.
 3. A method according toclaim 1 comprising co-administration, concomitantly or in sequence, of atherapeutically effective amount of a compound of formula I in free formor in pharmaceutically acceptable salt form, and a second drugsubstance, said second drug substance being an immunosuppressant,immunomodulatory, anti-inflammatory, antiproliferative or anti-diabeticdrug.
 4. A pharmaceutical composition comprising a compound of formula I

or a pharmaceutically acceptable salt thereof in association with apharmaceutically acceptable diluent or carrier therefore wherein R_(a)is H; CH₃; CH₂-CH₃; or isopropyl, R_(b) is H; halogen; C₁₋₆alkoxy; orC₁₋₆alkyl, and either I. R is a radical of formula (a)

wherein R₁ is piperazin-1-yl optionally substituted by CH₃ in position 3or 4; or 4,7-diaza-spiro [2.5] oct-7-yl; R₂ is Cl; Br; CF₃; or CH₃; andR₃ is H; CH₃; or CF₃; R₂ being other than CH₃ or Cl when R₃ is H, R_(a)is H or CH₃, R_(b) is H and R₁ is 4-methyl-1-piperazinyl; or II. R is aradical of formula (b)

wherein R₄is piperazin-1-yl substituted in positions 3 and/or 4 by CH₃;or 4,7-diaza-spiro [2.5] oct-7-yl; R_(a) being other than H or CH₃ whenR₄ is 4-methyl-1-piperazinyl; or II. R is a residue of formula (c)

wherein R₁₄ is 4,7-diaza-spiro [2.5] oct-7-yl; R₁₅ is halogen; CF₃; orCH₃; R₁₅ being other than CH₃ when R₁₆ is CH₃, R_(a) is H or CH₃, andR_(b) is H; and R₁₆ is H; CH_(3; CH) ₂-CH₃; or CF₃; R₁₆ being other thanH when R₁₅ is Cl, R_(a) is H or CH₃, and R_(b) is H; or IV. R is aradical of formula (d)

wherein R₈ is 1-piperazinyl, 3-methyl-piperazin-1-yl or4-benzyl-piperazin-1-yl; or V. R is a radical of formula (e)

wherein R₉ is 4,7-diaza-spiro [2.5] oct-7-yl; or 1-piperazinylsubstituted in position 3 by methyl or ethyl and optionally in position4 by methyl; or a pharmaceutically acceptable salt thereof.